The phenomenon of Surface Plasmon Resonance (SPR), first reported in 1968 (Otto, 1968, Zeitschrift Fur Physik 216: 398; Kretschmann and Raether, 1968, Zeitschrift Fur Naturforschrung Part A—Astrophysik, Physik, und Physikalische Chemie A 23: 2135), occurs due to the formation of surface plasmon polaritons, also known as surface plasmons, which are surface electromagnetic waves that propagate parallel to a metal thin film/dielectric interface and as such are very sensitive to changes at the interface (e.g. molecular adsorption). The angle of minimum reflectivity, known as the surface plasmon angle, depends strongly on the refractive index of the medium in contact with the metal surface, with an interaction depth that decays exponentially from the surface. This property has been exploited to form the basis of a highly sensitive and versatile sensing technology for measurements of surface binding in aqueous solutions. In an SPR experiment, a metal thin film is modified with molecules, such as DNA, capable of forming affinity interactions. Upon exposure of the surface to affinity partners, binding occurs that produces a change in reflectivity of the surface. A simple measurement of the amount of reflected light thus provides a sensitive and quantitative measure of surface binding.
An important advantage of SPR is that unlike fluorescence detection, no label is needed, eliminating the need for a labeling step on a molecule that may be easily damaged, and/or present only at a low concentration or purity. Surface plasmon resonance (SPR) imaging has demonstrated its ability to monitor interactions between biological moieties in real-time, without the aid of chemical labels such as fluorophores and radioisotopes. Currently gold surfaces modified with alkane thiol monolayers are used to monitor interactions via SPR. The technique of SPR imaging has proven its utility in monitoring DNA-DNA, DNA-protein, peptide-protein, small molecule-protein, protein-protein interactions; giving a wealth of information pertaining to enzyme kinetics, drug affinity studies, and DNA hybridization studies.
Although SPR is a powerful and widely used platform for the characterization of molecular interactions, like all technologies, it has limitations. For example, while SPR does detect binding, it does not provide much information on the nature of the binding molecule. This has tended to limit the application of the technology to the analysis of already known and purified molecules interacting with one another, and not allowed the technology to be of much use in the analysis of complex mixtures or for the discovery of previously unknown binding partners. In addition, the detection sensitivity is lower than that of some other methods, most notably evanescent wave fluorescence detection. SPR detection is similar to absorption spectrophotometry in that it measures small changes in a large signal (the reflected light). This limits its sensitivity compared to fluorescence in which small amounts of emitted light can be detected in the presence of little background signal, although achieving this in practice can be compromised by fluorescent contaminants and light scattering. Finally, the gold thin film generally employed for SPR measurements is physically fragile and the thiol-gold bonds used to place attachment chemistry on the gold surfaces are not stable to either UV irradiation (often employed in photochemical processes), or to a wide variety of moderate to harsh chemical conditions (e.g. acids, bases, oxidizers, reductants, etc.), limiting the sorts of chemistry that one can utilize on these substrates and compromising its utility for many applications. In addition, the gold surfaces typically employed in SPR experiments cannot be used in the creation of high-density microarrays in systems developed by Nimblegen and Affymetrix, which use UV-light photolithographic methods. Upon exposure to UV light gold-thiol bonds are cleaved, leaving the surface unusable for array fabrication. Thus the fabrication of a substrate that is both SPR-active and chemically robust is necessary to create a usable surface for label-free detection means in highly-parallel and multiplexed experiments, such as those increasingly used and relied upon in the areas of genomics, proteomics, and drug discovery. The present invention addresses these and related needs.